What buffer are you using?

Is your enzyme HF version? If not are you using the EcoRI buffer?

I found if I wasn't using the HF version and therefore used the EcoRI buffer (my enzymes are from NEB) I had similar problems ie extra band. However when I switched to the HF version (CutSmart buffer), I got better product. Hope this helps

I would use a plasmid as control for the digestion! This might rule out your enzyme conditions are fine! However, take into account the source of DNA might affect how the restriction proceeds! E.g. plasmid DNA is readily digested, while amplified DNA (PCR product) might be hard to digest. Try to EcoRI-HF!

Use a controlto check if the enzyme is not damaged and check if the EcoRI site is really there!

Let me know, and good luck

E

1- make sure you are using the right buffer

2- use more enzyme and/or for more time and at the right temperature--use twice the number of enzyme unites than waht you are using now

3- are you sure that every DNA molecule in your tube has the EcoRI site? (your DNA migh be a mixture of site plus, site minus and hybrid molecules!!

good luck

Thank you everyone for your answers. I will double-check all of the mentioned points above and let you know when the result comes out. 

Is there any chance for star activity by the use of DW?. I just found out that I use nonsterile DW for my enzyme reaction. Could it contribute to the incomplete digestion?

I think the problem may be due more to the small size of the fragment (in which the sticky ends naturally tend to reassemble). However, a problem in the functioning of the enzyme may be caused by glycerol (present in the storage buffer of the enzymes), which can interfere with the reaction, if it exceeds 10% of the total volume of the reaction mix.